Chemical analysis involving the detection and quantization of light occurs in a large variety of situations. One application of this need is the detection of analytes for the determination of the presence or amount of a particular analyte. In many assays for analytes, one is concerned with either absorption or emission (e.g., fluorescence or chemiluminescence) of light. In many situations, one irradiates a sample with light and then attempts to detect the effect of the sample on the transmitted or emitted light. In the case of emitted light resulting from irradiation, non-analyte molecules may also emit light resulting in a relatively large background noise, which results in the introduction of substantial error in the measurement of the effect of the sample on the light. There are also additional systematic errors which collectively contribute to the noise associated with the measurement.
The quality of chemical measurements involving light can be defined in terms of the ratio of a suitable measure of the optical signal from a sample due to the presence of analyte to the noise variation inherent within the signal. In general, efforts to augment this signal to noise (S/N) ratio have centered on improving the sensitivity of a measurement apparatus so as to reduce the "detection limit" associated with a particular analyte. The detection limit refers to the analyte concentration within a sample above which the signal attributable to the presence of analyte is such that a desired S/N ratio is achieved. In practice, this detection limit is ascertained by conducting an experimental procedure designed to elicit an optical signal related to analyte concentration. Specifically, data relating to signal and noise intensity is plotted in the form of a calibration curve for a range of analyte concentrations, thereby enabling straightforward determination of the detection limit.
The determination of concentration in unknown samples is effected by comparing the signal obtained experimentally from the unknown with the calibration curve. A typical unit of concentration in chemical measurements is moles/liter [i.e., Molarity (M)], where a mole is defined as Avogadro's number (6.0225.times.10.sup.23). Unfortunately, even the most sensitive conventional experimental techniques have detection limits on the order of about one femtomolar (fM), or nearly one billion analytes per liter.
Measurements in which concentration is determined by reference to a calibration curve may be characterized as being inherently "analog" rather than "digital". That is, a signal correlated with analyte concentration is initially produced by the measurement device. The calibration curve is then consulted to obtain an approximation of the analyte concentration. Since the calibration curve may be made continuous as a function of concentration, the concentration derived from the calibration curve will generally not be an integer. In contrast, measurement data in the digital domain are often embodied in binary (i.e., two-level) signals which unequivocally represent specific integers. Accordingly, a fundamental difference between analog and digital modes of measurement is that the addition of a single additional analyte to a sample analyzed using analog means cannot be unambiguously detected. Although dramatic improvements have been made in the accuracy of chemical measurements, such advancements have been based on the fundamentally analog concepts of increasing signal and reducing noise.
In molecular samples involving low levels of analyte concentration a digital measurement methodology would afford at least two advantages: (i) reference to a calibration curve would not be required, and (ii) the addition of a single additional molecule to a sample could conceivably be detected. Such a digital technique would be of utility in samples where the analyte concentration is sufficiently low that statistical noise accompanying each binary measurement value remains less than the difference between successive integers. Accordingly, it is an object of the present invention to provide an optical technique for determining low levels of analyte concentration by means of an intrinsically digital measurement scheme.